Method for measuring the binding sites for macrophage-derived inflammatory mediator (MIP-1α and MIP-1β)

ABSTRACT

An inflammatory cytokine is disclosed which has been isolated from cells that have been incubated with a stimulator material. The inflammatory cytokine comprises a protein that is capable of binding to heparin, inducing localized inflammation characterized by polymorphonuclear cell infiltration when administered subcutaneously and inducing in vitro polymorphonuclear cell chemokinesis, while lacking the ability to suppress the activity of the anabolic enzyme lipoprotein lipase, cause the cytotoxicity of cachectin/TNF-sensitive cells, stimulate the blastogenesis of endotoxin-resistant C3H/HeJ thymocytes, or induce the production of cachectin/TNF by primary thioglycollate-elicited mouse macrophage cells. A particular inflammatory cytokine MIP-1 has been isolated and has been found to comprise a peptide doublet of similar molecular weights of about 8,000 daltons, and to show a pI of about 4.6. The doublet has been resolved into its component peptides, MIP-1α and MIP-1β for which distinct cDNA&#39;s have been cloned and sequenced. Diagnostic and therapeutic utilities are proposed, and testing procedures, materials in kit form and pharmaceutical compositions are likewise set forth.

RELATED APPLICATIONS

The present application, is a Division of Application Ser. No.08/207,888 filed Mar. 7, 1994, now U.S. Pat. No. 5,616,688, which is acontinuation of Ser. No. 08/024,867, filed Mar. 1, 1993, now abandoned,which is a continuation of Ser. No. 07/902,532, filed Jun. 22, 1992 nowabandoned, which is a continuation of Ser. No. 07/238,937, filed Sep. 2,1988, now abandoned, which is a continuation of Ser. No. 07/104,827,filed Oct. 2, 1987, now abandoned, which is a continuation in part of07/766,852, filed Aug. 16, 1985 now abandoned, which is acontinuation-in-pars Ser. No. 06/414,098, filed Sep. 7, 1982, now U.S.Pat. No. 4,603,106, which is continuation-in-part of Ser. No.06/351,290, filed Feb. 22, 1982, now abandoned which is acontinuation-in-part of Ser. No. 06/299,932, filed Sep. 8, 1981, nowabandoned.

RELATED PUBLICATIONS

The Applicants are authors or co-authors of several articles directed tothe subject matter of the present invention. These articles are insupplementation to those articles listed in U.S. Pat. No. 4,603,106,which earlier articles are incorporated herein by reference. (1)[Applicants Cerami and Beutler are co-authors with J. Mahoney, N. LeTrang and P. Pekala] "Purification of Cachectin, a LipoproteinLipase-Suppressing Hormone Secreted By Endotoxin-Induced RAW 264.7Cells", J. EXP. MED. 161 at 984-995 (May, 1985); (2) [Applicantsco-authored with JAR. Mahoney, N. Le Trang, W. Vine, and Y. Ikeda]"Lipopolysaccharide-Treated RAW 264.7 Cells Produce a Mediator WhichInhibits Lipoprotein Lipase in 3T3-L1 Cells", J. IMMUNOL. 134 (3) at1673-1675 (March, 1985); (3) [Applicant Cerami a co-author with P. J.Hotez, N. Le Trang, and A. H. Fairlamb] "Lipoprotein Lipase Suppressionin 3T3-L1 Cells by a Haematoprotozoan-Induced Mediator From PeritonealExudate Cells." PARASITE IMMUNOL. (Oxf.) 6:203 (1984); (4) [ApplicantsCerami and Beutler co-authored with D. Greenwald, J. D. Hulmes, M. ChangY.-C.E. Pan, J. Mathison and R. Ulevitch] "Identity of Tumor NecrosisFactor and Macrophage-Secreted Factor Cachectin", NATURE 316:552-554,(1985); (5) [Applicants Cerami and Beutler co-authored with F. M. Torti,B. Dieckmann and G. M. Ringold] "A Macrophage Factor Inhibits AdipocyteGene Expression: An In Vitro Model of Cachexia" SCIENCE 229:867-869,(1985); (6) [Applicants Cerami and Beutler co-authored with I. W.Milsark] "Passive Immunization Against Cachectin/Tumor Necrosis Factor(TNF) Protects Mice From the Lethal Effect of Endotoxin", SCIENCE229:869-871, (1985); and (7) [Applicants Cerami and Beutler co-authoredwith S. D. Wolpe, G. Davatelis, B. Sherry, D. G. Hesse, H. T. Nguyen, L.I. Moldawer, C. F. Nathan and S. F. Lowry] "Macrophages Secrete A NovelHeparin-Binding Protein With Inflammatory And Neutrophil ChemokineticProperties", J. EXP. MED. 16F: 570-581 (1988). All of the above listedarticles are incorporated herein by reference.

The research leading to the present invention was funded in part bygrants from the National Institute of Health and the RockefellerFoundation.

BACKGROUND OF THE INVENTION

The present invention is generally directed to materials and associatedmethods for the analysis and treatment of the effects and correspondingoperation of invasive stimuli such as infection upon animal hosts, andin particular, is concerned with the identification of materials whichmay participate in the host response to such invasive stimuli.

Several common physiological and biochemical derangements have beenobserved in various mammalian hosts responding to a variety of invasivestimuli such as bacterial, viral or protozoal infection; tumors; orendotoxemia; as well as in idiopathic states. For example, theseresponses include fever, leukocytosis, hyperlipidemia, reduced foodintake (anorexia), reduced activity, wasting (cachexia), and othermodifications in muscle, white blood cell and liver metabolism. Inparticular, recent studies aimed at elucidating the biochemicalmechanisms of cachexia in rabbits infected with Trypanosoma brucei notedthat animals with a minimal parasite burden became moribund andexhibited an extreme hypertriglyceridemia, with a marked elevation ofplasma very low density lipoprotein (VLDL). See C. A. Rouser and A.Cerami, MOL. BIOCHEM. PARASITOL. 1:31-38 (1980). Thehypertriglyceridemic state was remarkable in view of he severe wastingdiathesis that accompanied this experimental infection. The elevation ofplasma VLDL was shown to result from a clearing defect, caused by a lossof peripheral tissue lipoprotein lipase (LPL) activity.

Reduced LPL activity has been observed by others, and it has been notedthat this condition has existed when the human body was in shock. See E.B. Man, et al., "The Lipids of Serum and Liver in Patients with HepaticDiseases", CLIN. INVEST. 24 at 623, et seq. (1945); See also John I.Gallin, et al., "Serum Lipids in Infection", N. ENGL. J. MED. 281 at1081-1086 (Nov. 13, 1969); D. Farstchi, et al., "Effects of ThreeBacterial Infections on Serum Lipids of Rabbits", J. BACTERIOL. 95 at1615, et seq. (1968); S. E. Grossberg, et al., "Hyperlipaemia FollowingViral Infection in the Chicken Embryo: A New Syndrome", NATURE (London)208 at 954, et seq. (1965); Robert L. Hirsch, et al., "Hyperlipidemia,Fatty Liver and Bromsulfophthalein Retention in Rabbits InjectedIntravenously with Bacterial Endotoxin", J. LIPID. RES. 5 at 563-568(1964); and Osamu Sakaguchi, et al., "Alternations of Lipid Metabolismin Mice Injected With Endotoxins", MICROBIOL. IMMUNOL. 23 (2) at 71-85(1979); R. F. Kampschmidt, "The Activity of Partially PurifiedLeukocytic Endogenous Mediator in Endotoxin Resistant C3H/HeJ Mice", J.LAB. CLIN. MED. 95 at 616, et seq. (1980); and Ralph F. Kampschmidt,"Leukocytic Endogenous Mediator", J. RET. SOC. 23 (4) at 287-297 (1978).

Additionally, publications are known by the Applicants that discuss theidentification and existence of "mediators" that appear to be involvedin the host response to infection; and in particular, the followingarticles, the texts of which are incorporated herein by reference, arelisted: Sipe, J. D., et al., J. EXP. MED., 150:597-606 (1979); andBarney, C. C., et al., LIPTON, J. M. (Ed.), FEVER: INTERNATIONALSYMPOSIUM, Dallas, Tex., Apr. 11-12, 1979 XII+263P. Raven Press: NewYork, Illus. ISBN 0-89004-451-1 (08877), 0 (0), pp.111-122 (1980); andDinarello, C. A., "Human Leukocytic Pyrogen: Purification andDevelopment of a Radioimmunoassay", PROC. NATL. ACAD. SCI. USA, 74(10)at 4624-4627 (October, 1977). All of the factors identified andinvestigated by each of the authors in the above noted articles and thearticles authored or co-authored by Kampschmidt have been determined tocomprise a single grouping of factors which has been identified asinterleukin-1 (IL-1). This determination has been documented in anarticle by Charles A. Dinarello, published in REVIEWS OF INFECTIOUSDISEASES, at Volume 6, No. 1 (January-February, 1984), the text of whichis also incorporated herein by reference.

A similar deficiency of LPL activity was noted by Applicants in C3H/HeNmice after administration of Escherichia coli lipopolysaccharide (LPS).In contrast, the loss of LPL activity was not demonstrable in C3H/HeJmice, which are genetically resistant to LPS. This resistance toendotoxin-induced LPL deficiency could be circumvented by theadministration of serum obtained from endotoxin-sensitive animals thathad been injected with LPS two hours previously. Similarly, resistancecould be overcome by injecting conditioned medium fromendotoxin-stimulated thioglycollate-elicited peritoneal macrophagesobtained from sensitive mice. These findings were set forth in fulldetail in application Ser. No. 414,098, now U.S. Pat. No. 4,603,106, thedisclosure of which is incorporated herein by reference.

The above work was prompted by the Applicants' belief that a "mediator"or "mediators" existed and were suspected to have a significant effectupon general metabolic activity of energy storage cells in the animalhost. It was suspected that such "mediators" exerted a depressive effectupon the activity of certain anabolic enzymes, whose reduced activitywas observed, for instance, where the host enters the condition known asshock, as in response to invasion. Resultingly, the relationship of themediator produced by endotoxin-stimulated peritoneal mouse exudatecells, upon endotoxin-sensitive and endotoxin-insensitive mice alike,and the development through such investigation of a reagent for themeasurement of anabolic enzyme activity was set forth in first filedabandoned application Ser. No. 299,932, incorporated herein byreference.

Further investigation of this system was made in conjunction with the3T3-L1 "preadipocyte" model system, and the corresponding development ofmethods and associated materials for the development of antibodies tothe "mediator" and other diagnostic procedures was then set forth inapplication Ser. No. 351,290, also incorporated herein by reference andnow abandoned. Thereafter, in subsequent application Ser. No. 414,098,now U.S. Pat. No. 4,603,106, the Applicants established that themediator substance that they had derived from the endotoxin stimulationof macrophage cells exhibited the activities of suppressing the anabolicenzymes lipoprotein lipase, acetyl Coenzyme A Carboxylase and fatty acidsynthetase, and further, inhibited the growth and differentiation oferythroid-committed cells.

Additional work set forth in articles (1) and (4) by Beutler et al., andapplication Ser. No. 766,852 now abandoned, the disclosure of which isincorporated herein by reference, has resulted in the discovery that theearlier identified mediator substance contained a further proteincomponent which possesses a number of activities, which distinguished itfrom both the mediator substance and the other factors identified in theart and known as Interleukin 1 and Interleukin 2. Further work set forthin article (f) by Beutler et al. and parent application Ser. No. 104,827now abandoned, the disclosure of which is incorporated herein byreference, established the presence of an additional factor (MIP-1) inthe mediator substance which demonstrates a distinguishable profile ofactivities.

Since that time, MIP-1 has been resolved into component peptides and theN-terminal sequences of two such peptides, now referred to as MIP-1α andMIP-1β, have been compared. Accordingly, the present application isdirected to the newly discovered peptide isolates and the activities ofMIP-1, and the applications both diagnostic and therapeutic to whichthese isolates may be put.

SUMMARY OF THE INVENTION

In accordance with the first aspect of the present invention, theinflammatory cytokine that is the newly discovered isolate of themediator substance is disclosed, and comprises a protein that has beenpurified and is anionic under physiological conditions. The inflammatorycytokine of the present invention exhibits the ability to bind toheparin, even at high salt concentrations, to induce localizedinflammation characterized by polymorphonuclear cell infiltration whenadministered subcutaneously and to induce in vitro polymorphonuclearcell chemokinesis. The present inflammatory cytokine however, lackscertain activities common to other factors that have been isolated fromthe mediator substance disclosed in U.S. Pat. No. 4,603,106.

In particular, the present inflammatory cytokine lacks the ability tosuppress the activity of the anabolic enzyme lipoprotein lipase (LPL),and is unable to cause the cytotoxity of cachectin/TNF-sensitive L929cells, to stimulate the blastogenesis of endotoxin-resistant C3H/HeJthymocytes, or to induce the production of cachectin/TNF by primarythioglycollate-elicited mouse macrophage cells. These lattercharacteristics all absent from the present inflammatory cytokine areexhibited by the known factors cachectin/TNF and interleukin-1 (IL-1),and thereby distinguish the present inflammatory cytokine therefrom.

Of the affirmative activities exhibited, the ability to bind to heparinand to induce localized inflammation characterized by polymorphonuclear(PMN) cell infiltration appear most significant. Accordingly, while theexact role that the present isolate plays in the cascade of reactions tohost invasion is as yet ill defined, its participation in theelicitation of certain of the activities and conditions associated withmobilization against host invasion is clear. Accordingly, theinflammatory cytokine possesses the potential for use as a diagnostictool to identify and perhaps differentiate between various stimuliwhether invasive or idiopathic, by the activation of the presentinflammatory cytokine that such stimuli may promote.

The present inflammatory cytokine was initially partially identified andcharacterized and found to contain certain polypeptide segments definingan apparent molecular weight of approximately 8,000 daltons asdetermined on polyacrylamide gel electrophoresis in the presence ofsodium dodecyl sulphate (SDS-PAGE) with the tendency to form aggregatesof high molecular weight greater than about 10⁶ daltons in low saltbuffers. In particular, the present inflammatory cytokine has been notedto form aggregates greater than 2×10⁶ daltons as assessed by gelfiltration. Partial N-terminal amino acid sequence data as depicted inFIG. 2 herein, reveals no significant homology with any previouslydescribed proteins. By known cDNA cloning techniques based upon thepartial peptide sequences determined, full translated peptide sequenceswere achieved, which have been found to possess molecular weights ofapproximately 8 kilodaltons. MIP-1 has been shown to have a pI ofapproximately 4.6 by chromatofocusing.

Further properties of the present inflammatory cytokine include itsability to induce fever in rabbits, and to induce superoxide formationor a respiratory burst in human neutrophils in vitro. These propertiesare illustrated in the data presented herein.

The present invention is also directed to the resolution of purifiednative MIP-1 which migrates as a doublet on SDS-PAGE with nearlyidentical apparent component molecular weights of about 8000 daltons.Chromatography on hydroxylapatite in the presence of SDS successfullyseparated the two components. Partial N-terminal sequence analysis ofeach component revealed that the two proteins are very similar in theirN-terminal sequence but differ in the presence and positions of certainamino acids. In particular, the protein corresponding to the lowermolecular weight band on SDS-PAGE (now termed "MIP-1a") had a partialN-terminal amino acid sequence identical to the major sequence observedand shown in FIG. 3 herein, whereas the higher molecular weight band(termed "MIP-1β") yielded an N-terminal sequence identical to thesequence obtained after substituting the minor amino acids at theirrespective positions. cDNA'S for both MIP-1α and MIP-1β have beencloned, allowing for the determination of the complete amino acidsequence of each of the peptide components, which sequences are depictedin FIGS. 10 and 15, respectively.

The cDNA for MIP-1α predicts a mature protein of 69 amino acids inlength with a predicted molecular mass of 7,889. There are no apparentsites for N-glycosylation. The cDNA for MIP-1β predicts a mature proteinalso of 69 amino acids in length with a predicted molecular mass of7,832 daltons. There is one potential N-glycosylation site (Asn-Pro-Ser)at positions 53 to 55.

As mentioned earlier, the present inflammatory cytokine may be preparedby the stimulation of animal cells with a material such as mightaccompany an invasive stimulus. In particular, a sample of macrophagecells which may be derived from a variety of sources may be incubatedwith a stimulator material such as endotoxin or trypanosomes, to producethe mediator substance disclosed in U.S. Pat. No. 4,613,106. Suchincubation may take place for a period of time of up to twenty hours,and exact time limits will vary with the particular cells selected forincubation.

Following such incubation, the medium may be appropriately treated as bycentrifuging, to remove a supernatant containing the crude mediatorsubstance. The mediator substance may then be further treated as byfiltration or precipitation. Thereafter, the crude mediator substancemay be subjected to a series of known isolation techniques, whereuponthe inflammatory cytokine may be recovered. The present inventionnaturally contemplates alternate means for preparation of theinflammatory cytokine, including where applicable known geneticreplicative techniques, and the invention is accordingly intended tocover such synthetic preparations within its scope.

As noted above, the present invention also includes the identificationof the purified peptide components of the present cytokine that exhibitin combination the above noted activities and characteristics, thatdisplay the amino acid sequences set forth below and in FIGS. 10 and 15,as determined in mice.

    MIP-1α                                                                  ALA PRO TYR GLY ALA ASP THR PRO THR ALA CYS CYS                               PHE SER TYR SER ARG LYS ILE PRO ARG GLN PHE ILE                               VAL ASP TYR PHE GLU THR SER SER LEU CYS SER GLN                               PRO GLY VAL ILE PHE LEU THR LYS ARG ASN ARG GLN                               ILE CYS ALA ASP SER LYS GLU THR TRP VAL GLN GLU                               TYR ILE THR ASP LEU GLU LEU ASN ALA                                           MIP-1β                                                                   ALA PRO MET GLY SER ASP PRO PRO THR SER CYS CYS                               PHE SER TYR THR SER ARG GLN LEU HIS ARG SER PHE                               VAL MET ASP TYR TYR GLU THR SER SER LEU CYS SER                               LYS PRO ALA VAL VAL PHE LEU THR LYS ARG GLY ARG                               GLN ILE CYS ALA ASN PRO SER GLU PRO TRP VAL THR                               GLU TYR MET SER ASP LEU GLU LEU ASN                                       

As stated earlier, the foregoing sequence bears no striking similarityto any of the known mediator factors and accordingly establishes thatthe present inflammatory cytokine is distinguishable therefrom. Therecent isolation of the above amino acid sequences and the developmentof the mRNA sequence have facilitated the reproduction of this cytokineby conventional recombinant genetic techniques.

The invention further includes methods for identifying idiopathic orinvasive stimuli on the basis of their ability to induce the presentinflammatory cytokine or the activities that it affects. In particular,such stimuli could be identified and detected by their ability to inducemediators which bind to heparin and induce localized inflammation withneutrophil infiltration and chemokinesis. In this method, macrophagecells derived for example, from the RAW 264.7 cell line could beinoculated with a number of known stimulator materials such asendotoxin, trypanosomes or the like, as a control, while parallelcellular samples could be inoculated with an extract of material fromthe presumed situs of the infective stimulus. All samples couldthereafter be incubated in accordance with the methods described above,and thereafter subjected to the sequence of separation techniques alsodefined, whereupon testing of the resulting isolates derived from thecontrol and unknown samples could be compared to determine whether theinflammatory cytokine, if any, developed is identical or even similar.

Alternatively, altered mRNA levels of the inflammatory cytokine could bedetected by techniques used in the art that make use of cDNA sequencesdisclosed herein.

In similar fashion, an assay system for screening of potential drugseffective to either mimic or counteract the inflammatory cytokine may beprepared. In one instance, the test drug could be administered to astimulated macrophage sample to determine its effect upon the productionof the inflammatory cytokine. In an alternate procedure, theinflammatory cytokine may be introduced into a cellular test system inwhich the cytokine is known to be active, and the prospective drug mayalso be introduced to the same cell culture and the culture maythereafter be examined to observe any changes in the activity of theinflammatory cytokine in comparison with the addition of the prospectivedrug alone, or the effect of added quantities of the known inflammatorycytokine.

The present invention also relates to a method for determining thepresence of stimulated, spontaneous, or idiopathic pathological statesin mammals, by measuring the activity and presence of the inflammatorycytokine of the present invention. More particularly, the activity ofthe inflammatory cytokine may be followed directly by the assaytechniques discussed later on, through the use of an appropriatelylabeled quantity of the cytokine. Alternately, the cytokine can be usedto raise binding partners or antibodies that could in turn, be labeledand introduced into a medium such as serum, to test for the presence ofinflammatory cytokine therein, and to thereby assess the state of thehost from which the medium was drawn.

Thus, both the inflammatory cytokine and any antibodies that may beraised thereto, are capable of use in connection with various diagnostictechniques, including immunoassays, such as a radioimmunoassay, usingfor example, an antibody to the inflammatory cytokine that has beenlabeled by either radioactive addition, reduction with sodiumborohydride, or radioiodination.

In an exemplary immunoassay, a control quantity of the inflammatorycytokine, its antibody, or the like may be prepared and labeled with anenzyme, a specific binding partner and/or a radioactive element, and maythen be introduced into a blood sample of a mammal believed to beundergoing invasion. After the labeled material or its bindingpartner(s) has had an opportunity to react with sites within the sample,the resulting mass may be examined by known techniques, which may varywith the nature of the label attached.

In the instance where a radioactive label, such as the isotopes ¹⁴ C,¹³¹ I, ³ H, ¹²⁵ I and ³⁵ S are used, known currently available countingprocedures may be utilized. In the instance where the label is anenzyme, detection may be accomplished by any of the presently utilizedcolorimetric, spectrophotometric, fluorospectrophotometric or gasometrictechniques known in the art.

The present invention includes an assay system which may be prepared inthe form of a test kit for the quantitative analysis of the extent ofthe presence of the inflammatory cytokine. The system or test kit maycomprise a labeled component prepared by one of the radioactive and/orenzymatic techniques discussed herein, coupling a label to theinflammatory cytokine; and one or more additional immunochemicalreagents, at least one of which is a free or immobilized ligand, capableeither of binding with the labeled component, its binding partner, oneof the components to be determined or their binding partner(s).

In a further embodiment, the present invention relates to certaintherapeutic methods which would be based upon the activity of theinflammatory cytokine, antibodies to the inflammatory cytokine, or uponother agents or drugs determined to possess the same or an antagonisticactivity. A first therapeutic method is associated with the preventionof the manifestations of the activities of the inflammatory cytokine inmammals, such as inflammation and fever, and comprises administeringeither an antibody to the cytokine, an agent capable of modulating theproduction and/or activity of the cytokine, or an agent not an antibodyto the cytokine that is capable of acting as an antagonist to thecytokine, either individually or in mixture with each other in an amounteffective to prevent the development of those conditions in the host.

More specifically, the therapeutic method generally referred to hereincould include the method for the treatment of inflammation and fever bythe administration of pharmaceutical compositions that may compriseeffective quantities of antibodies to the inflammatory cytokine, orother equally effective drugs developed, for instance, by a drugscreening assay prepared and used in accordance with a further aspect ofthe present invention. A variant embodiment of this therapeutic methodcould include initially detecting the presence and activity of theinflammatory cytokine and thereafter admininstering the appropriatepharmaceutical composition.

A second therapeutic method seeks to take advantage of the inflammatoryactivity of the cytokine and in particular, its ability to cause themovement and mobilization of neutrophils in response to invasive stimulisuch as infection. Accordingly, the inflammatory cytokine may beprepared in a suitable formulation for administration to the situs ofinfection which for example, may develop where tissue trauma hasoccurred. In such instance, the inflammatory cytokine prepared in asterile solution and delivered to the trauma or wound as part of anirrigation fluid or by direct dosage such as, in a pharmaceuticalcomposition, the latter course of administration contemplating topicaland parenteral routes. Naturally, the inflammatory cytokine may be usedto raise equally effective agents or drugs by known methods that maythen be formulated into pharmaceutical compositions suitable foradministration in the same manner and for the same purpose as for theinflammatory cytokine itself.

Accordingly, it is a principal object of the present invention toprovide an inflammatory cytokine in purified form that exhibits certaincharacteristics and activities associated with the host response toinvasive stimuli in mammals.

It is a further object of the present invention to provide a method forthe preparation of the inflammatory cytokine.

It is a further object of the present invention to provide a method fordetecting the presence of the inflammatory cytokine in mammals in whichinvasive, spontaneous, or idiopathic pathological states such asinfection are suspected to be present.

It is a further object of the present invention to provide a method andassociated assay system for screening substances such as drugs, agentsand the like, potentially effective in either mimicking the activity orcombating the adverse affects of the inflammatory cytokine in mammals.

It is a still further object of the present invention to provide amethod for the treatment of mammals to control the amount or activity ofthe inflammatory cytokine, so as to alter the consequences of suchpresence or activity.

It is a still further object of the present invention to provide amethod for the treatment of mammals to promote the amount or activity ofthe inflammatory cytokine, so as to treat or avert the adverseconsequences of invasive, spontaneous or idiopathic pathological states.

It is a still further object of the present invention to provideharmaceutical compositions for use in therapeutic methods which compriseor are based upon the inflammatory cytokines or their bindingpartner(s), or upon agents or drugs that control the production, or thatmimic or antagonize the activities of the inflammatory cytokine.

Other objects and advantages will become apparent to those skilled inthe art from a review of the ensuing description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a combined graphical and electrophoretic gel depiction of thepreparation and recovery of the inflammatory cytokine from a supernatantof RAW 264.7 cells. Fractionation of the concentrated, diafiltratedsupernatant from RAW 264.7 was performed on Mono Q. Two liters ofsupernatant (mediator substance) were concentrated twenty times anddiafiltrated against six liters of 20 mM Tris-HCl, pH 8.0. Theconcentrated supernatant was applied to Mono Q and eluted with a lineargradient of 0 to 1 M NaCl in the same buffer. MIP-1 (*) eluted slightlybefore cachectin/TNF (**) at approximately 0.37 M NaCl. The insert showsa 10-15% SDS-PAGE gel to which 50 μl from the indicated fractions havebeen applied.

FIG. 2 is a combined graphical and electrophoretic gel depiction offurther gel separation and purification of the present inflammatorycytokine. Peak MIP-1 containing fractions from Mono Q were concentratedto 200 μl and applied to a Superose 12 column equilibrated with 100 mMammonium acetate. MIP-1 (*) eluted in the void volume, well separatedfrom cachectin/TNF (**). The insert shows a 10-18% SDS-PAGE gel to which50 μl aliquots from the indicated fractions have been applied. Thedagger indicates purified MIP-1 used as a marker.

FIG. 3 depicts a partial amino acid sequence of the first 31 positionsof the inflammatory cytokine of the present invention. Residues inparentheses indicate positions at which "minor" residues werereproducibly obtained in different sequence runs on different batches ofmaterial.

FIG. 4 is a combined graphical and electrophoretic gel depiction of thebinding and elution of the inflammatory cytokine of the presentinvention to heparin. Two ml of 12-fold concentrated and diafiltratedLPS-stimulated RAW 264.7 supernatant were applied to a heparin-Sepharosecolumn and eluted with a linear gradient of 0 to 2 M NaCl in 0.02 MTris-HCl buffer, pH 7.8. Two major peaks were observed; MIP-1 (*) elutedin the second peak, corresponding to 0.6-0.75 M NaCl. The insert shows a10-18% acrylamide gradient SDS-PAGE gel to which 50 82 l aliquots fromthe indicated fractions have been applied.

FIG. 5 is a photomicrograph of C3H/HeJ footpads fixed 4 hours followingsubcutaneous injection of the inflammatory cytokine of the presentinvention with controls and comparative injections of cachectin/TNF.Tissues were fixed in buffered formalin and stained withhematoxylin-eosin. Magnification is 400×. Plate A--Sham injectionsreflecting normal histology. Plate B--100 ng MIP-1: moderateinfiltration of neutrophils and mast cells. Plate C--100 ngcachectin/TNF: moderate infiltration of neutrophils. Plate D--10⁻¹⁰moles FMLP: extensive infiltration with some focal necrosis.

FIGS. 6 graphically depicts the results of tests for the induction ofneutrophil chemokinesis by the inflammatory cytokine of the presentinvention. Data represent the mean percent of the control migratoryresponse of five experiments. Values are presented as the percentincrease in neutrophil migration relative to the Gey's basic saltsolution negative control (0%) and the 10⁻⁸ M fMLP positive control(100%). *=concentrations of MIP-1 showing significant increases in thepercent chemokinetic response versus GBSS alone (p<0.0l by ANOVA).

FIG. 7 is a graphical depiction of the results of tests for the abilityof the inflammatory cytokine to stimulate H₂ O₂ release by humanneutrophils (PMN'S). Human PMN'S incubated in serum-coated microtestplate wells were treated at time 0 with PMA (100 ng/ml) (◯), recombinantcachectin/TNF (10 ng/ml) (Δ), MIP-1 (1000 ng/ml) (∘) or an equivalentvolume of buffer alone (control) (▴). H₂ O₂ release was monitored overthe next 4.5 hours (2 hours for cachectin/TNF). Values are means±SEM fortriplicates in one of 4 similar experiments.

FIG. 8 is a depiction of the amino acid sequence of the inflammatorycytokine MIP-1 of the present invention as initially recovered frommice.

FIG. 9 depicts 512-fold degeneracy probe pools used in the cDNA cloningof MIP-1α. An asterisk below a base indicates a constant base changebetween the two-probe pools.

FIG. 10 depicts the complete nucleotide sequence of a cDNA clone forMIP-1α. The underlined sequence indicates the complementary sequence ofthe oligonucleotide used in primer extension experiments. The predictedtranslated molecular weight of the precursor peptide is 10,346. Themature peptide sequence, starting at position one, is 69 amino acids inlength and has a predicted molecular weight of 7,889.

FIG. 11 is an autoradiogram of a northern blot of total RNA from RAW264.7 cells, a transformed murine macrophage cell line. The cells werestimulated with LPS at 2 μg/ml in serum-free medium for 6 hours. Controlcells were given serum-free medium with no added LPS for 6 hours. Thetotal amount of RNA loaded in each lane is indicated on the figure.

FIG. 12 is a depiction of a time-course study of RAW 264.7 mRNAinduction by LPS. The time course is measured in hours. All three probeswere plasmid cDNA clones labeled with α-[³² p] dCTP.

FIG. 13 is a graphical depiction of the fractionation of MIP-1 intocomponent peptides by SDS-hydroxylapatite chromatography. MIP-1 (500 μg)was applied to SDS-hydroxylapatite and fractionated as described. 0.5 mlfractions were collected, analyzed by SDS-PAGE, and pooled as indicatedby underline.

FIG. 14 depicts N-terminal amino acid sequences: (A) The N-terminalamino acid sequence of purified MIP-1α; (B) The N-terminal amino acidsequence of purified MIP-1β, the residues underlined (2-8) were thoseused to construct an oligonucleotide probe pool; (C) The originalN-terminal amino acid consensus sequence reported for purified nativeMIP-1. Amino acid residues corresponding to the minor residues are givenin parenthesis.

FIG. 15 depicts the complete nucleotide sequence of the cDNA clone forMIP-1β. The predicted translated molecular weight is 10,169 daltons. Themature protein sequence, starting at position one, is 69 amino acids inlength and has a predicted molecular weight of 6993 daltons.

FIG. 16 is a graphical depiction of estimated local 20 hydropathicityalong the length of MIP-1α (---) and MIP-1β (--,--) calculated by anadaptation of the algorithm of Hopp and Woods.

DETAILED DESCRIPTION

In its primary aspect, the present invention concerns the isolation andidentification of a particular factor hereinafter referred to asinflammatory cytokine or macrophage inflammatory protein, MIP-1, thathas been found to be present in or secreted by macrophages or macrophagecell lines that are stimulated by materials referred to herein asstimulator materials, that characteristically accompany an invasivestimulus, such as bacteria, viruses, certain tumors, protozoa and othertoxins such as endotoxin, or an idiopathic state. The present inventionalso pertains to the resolution of MIP-1 into its component peptides,MIP-1α and MIP-1β. As with the mediator substance disclosed in U.S. Pat.No. 4,603,106, the present inflammatory cytokine, which has beendetermined to be a component of the former mediator substance, appearsto be capable of causing certain conditions such as inflammation todevelop in the tissues of a mammal, which reflect the reaction of amammal in a stimulated or spontaneous pathological state.

In particular, the inflammatory cytokine appears to be capable ofinducing localized inflammation when administered subcutaneously whichinflammation is characterized by polymorphonuclear cell infiltration.Also, the cytokine causes in vitro polymorphonuclear cell chemokinesis,which conditions are reflective of the influence of a cytokine involvedin mobilization by the mammalian host against an invasive stimulus.While the full and exact role played by the present inflammatorycytokine is unclear, it is theorized that the cytokine in conjunctionwith other factors previously identified and those yet to be elucidated,functions as part of a communication system between the immune system ofthe host and other body tissues and organs.

The ability of the present inflammatory cytokine to bind to heparin gaverise to the consideration that the cytokine might correspond to certainheparin-binding growth factors such as FGF or PDGF. However, dataindicating that the inflammatory cytokine is not mitogenic for smoothmuscle cells suggests a distinction from these known growth factors.Accordingly, what is certain at this time, is that the cytokine of thepresent invention participates in the development of the inflammatoryresponse that is known to to be a part of host responses such as toinvasion.

As indicated earlier, the present inflammatory cytokine has beenconfirmed to comprise a protein that possesses an isoelectric point (pI)of approximately 4.6. The inflammatory cytokine can be isolated as adoublet with nearly identical subunit molecular weights of approximately8,000 daltons and may form multimers of various molecular weights up toand exceeding 2×10⁶ daltons as assessed by the results of theinvestigations that led to its isolation, which are set forth in theExample below. These investigations likewise identified the N-terminalpeptide sequences, and the full sequence thereafter developed by knowngenetic replicative techniques confirm that the specific peptidesequences of the present inflammatory cytokine differ from that of otherknown mediator factors. Accordingly, both structural and functionaldistinctions between the present inflammatory cytokine and the knownmediator factors of the prior art exists as is confirmed by the data setforth in the Example.

More particularly, the inflammatory cytokine of the present inventionpossesses certain other characteristics in conjunction with thoseoutlined above, in that it is capable of binding to heparin at high saltconcentrations, e.g. approximately 0.7 M. A further characteristic ofthe present inflammatory cytokine is that it is anionic underphysiological conditions. The cytokine is also distinctive in thoseactivities that it lacks, such as the inability to suppress the anabolicenzyme lipoprotein lipase (LPL), to cause the cytotoxicity ofcachectin/TNF-sensitive L929 cells, stimulate the blastogenesis ofendotoxin-resistant C3H/HeJ thymocytes or to induce the production ofcachectin/TNF by primary thioglycollate-elicited mouse macrophage cells.All of these latter activities are those shared by the other knownmacrophage-derived mediator factors whose general characteristics andactivities have identified them as participants in the host response toinvasion. This accordingly distinguishes the present inflammatorycytokine from those known factors and confirms in conjunction with thecDNA and protein sequencing data presented herein, that the presentinflammatory cytokine is indeed distinct from the othermacrophage-derived mediator factors.

The inflammatory cytokine in accordance with the present invention wasisolated and analyzed in mice as set forth in the Example herein.Further work of cloning and sequencing the cDNA of the message for thedistinct polypeptides of the inflammatory cytokine performed after thecompletion of the experiments set forth in the Example has resulted inthe elucidation of the complete sequence of the peptides MIP-1α andMIP-1β, and their sequences are presented herein in FIGS. 10 and 15,respectively. It has therefore been determined that the purifiedinflammatory protein is defined by two sequences each of 69 amino acids.

A cDNA library was prepared from endotoxin-stimulated RAW 264.7 cellsand was screened using two synthetic oligonucleotide pools based on the"major" partial N-terminal amino acid sequence of MIP-1. The cDNA socloned is shown to correspond to the peptide chain now known as MIP-1α.The cDNA for MIP-1β was cloned using a similar strategy, although herethe oligonucleotide pool used was derived from a portion of the moleculein which three of the seven amino acid residues are different from thecorresponding residues in MIP-1α.

The cDNA for MIP-1α predicts a mature protein of 69 amino acids inlength with a predicted molecular mass of 7,889. There are no apparentsites for N-glycosylation. The cDNA for MIP-1β predicts a mature proteinalso 69 amino acids in length with a predicted molecular mass of 7,832daltons in which there is one potential N-glycosylation site(Asn-Pro-Ser) at position 53-55. As MIP-1β migrates on SDS-PAGE as aslightly larger molecule than MIP-1α, it is possible that it isglycosylated. It is known that proline at position X in theAsn-X-Ser,Thr signal for N-linked glycosylation results in impaired orabsent glycosylation at that site. This may account for the relativelysmall difference in molecular mass between MIP-1α and MIP-1β should thelatter be glycosylated.

Experiments are now under way to express the recombinant form of thisdisclosed protein. Human inflammatory cytokine MIP-1 is presumablysimilar to mouse MIP-1, since the mouse MIP-1 has an effect upon humanneutrophils. As disclosed herein, this activity of the inflammatorycytokine may be harnessed by administering the inflammatory cytokine tothe situs of tissue infection to promote the delivery of neutrophils tothat location.

The genetic replication of the inflammatory cytokine involves many ofthe general principles of recombinant technology that are well known inthe art, and accordingly a detailed presentation of such techniques isnot deemed to be necessary and is not presented herein. The presentinvention, however, contemplates the preparation of the presentinflammatory cytokine, and in particular the material having the aminoacid sequences set forth in FIGS. 10 and 15, by known recombinanttechniques. The preparation of the inflammatory cytokine was discussedin brief earlier herein, and is confirmed to be capable in one aspect ofproceeding by the initiation of the incubation of any of a variety ofcells with stimulator materials such as from invasive stimuli. Inparticular, the cell line RAW 264.7 may be utilized to initiate theproduction of the mediator substance from which the inflammatorycytokine may be isolated. The murine macrophage cell line RAW 264.7 hasfacilitated the isolation of the inflammatory cytokine in quantitieslarge enough to permit analysis and purification. Naturally, other celllines or other sources for the development of either the material fromwhich the inflammatory cytokine is thereafter isolated (mediatorsubstance), or the inflammatory cytokine or its constituent peptides,are contemplated herein and the present invention is accordingly notlimited. Thus, alternate means such as by genetic replication arecontemplated herein in accordance with the present invention.

As discussed earlier, the inflammatory cytokine, its constituentpeptides, their binding partner(s) or other ligands or agents exhibitingeither mimicry or antagonism to the cytokine or control over itsproduction, may be prepared in pharmaceutical compositions, with asuitable carrier and at a strength effective for administration byvarious means to a patient having a tissue infection or otherpathological derangement, for the treatment thereof. A variety ofadministrative techniques may be utilized, among them topicalapplications as in ointments or on surgical and other topical appliancessuch as, surgical sponges, bandages, gauze pads, and the like. Also,such compositions may be administered by parenteral techniques such assubcutaneous, intravenous and intraperitoneal injections, includingdelivery in an irrigation fluid used to wash body wound areas,catheterization and the like. Average quantities of the inflammatorycytokine and/or the above recited related agents may vary and inparticular should be based upon the recommendations and prescription ofa qualified physician or veterinarian.

As stated above and as indicated earlier, antibodies and drugs thatmodulate the production or activity of the inflammatory cytokine maypossess certain therapeutic applications and may thus be utilized forthe purpose of treating the effects attributable to the action of theinflammatory cytokine, such as inflammation and fever. In particular,the inflammatory cytokine may be used to produce antibodies to itself ina variety of mammals, by known techniques such as the hybridomatechnique utilizing, for example, fused mouse spleen lymphocytes andmyeloma cells. The resulting antibodies could then be prepared in asuitable pharmaceutical composition and administered to avert or treatthe undesired condition. The exact quantities, intervals ofadministration and administrative techniques respecting suchpharmaceutical compositions may vary in accordance with those known inthe medical arts, and upon the specific instruction of a qualifiedphysician or veterinarian.

The present invention also relates to a variety of diagnosticapplications, including methods for determining the presence of invasivestimuli by reference to their ability to elicit the activities which areaffected by the present inflammatory cytokine. As mentioned earlier, theinflammatory cytokine can be used to produce antibodies to itself by avariety of known techniques, and such antibodies could then be isolatedand utilized as in tests for the presence of the inflammatory cytokinein suspect mammals.

Antibody(ies) to the peptides of the inflammatory cytokine can beproduced and isolated by standard methods including the well knownhybridoma techniques. For convenience, the antibody(ies) to theinflammatory cytokines will be referred to herein as Ab₁ andantibody(ies) raised in another species as Ab₂.

The presence of inflammatory cytokine activity in mammals can beascertained by the usual immunological procedures applicable to suchdeterminations. A number of useful procedures are known. Three suchprocedures which are especially useful utilize either the inflammatorycytokine labeled with a detectable label, antibody Ab₁ labeled with adetectable label, or antibody Ab₂ labeled with a detectable label. Theprocedures may be summarized by the following equations wherein theasterisk indicates that the particle is labeled, and "Cyt" stands forthe inflammatory cytokine:

A. Cyt*+Ab₁ =Cyt*Ab₁

B. Cyt+Ab*=CytAb₁ *

C. Cyt+Ab₁ +Ab₂ *=CytAb₁ Ab₂ *

The procedures and their application are all familiar to those skilledin the art and are presented herein as illustrative and not restrictiveof procedures that may be utilized within the scope of the presentinvention. The "competitive" procedure, Procedure A, is described inU.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure C, the "sandwich"procedure, is described in U.S. Pat. Nos. RE 31,006 and 4,016,043. Stillother procedures are known such as the "double antibody", or "DASP"procedure.

In each instance, the inflammatory cytokine forms complexes with one ormore antibody(ies) or binding partners and one member of the complex islabeled with a detectable label. The fact that a complex has formed and,if desired, the amount thereof, can be determined by known methodsapplicable to the detection of labels.

It will be seen from the above, that a characteristic property of Ab₂ isthat it will react with Ab₁. This is because antibodies raised in onemammalian species have been used in another species as an antigen toraise antibodies such as Ab₂. For example, Ab₂ may be raised in goatsusing rabbit antibodies as antigens. Ab₂ therefore would be anti-rabbitantibody raised in goats. For purposes of this description and claims,Ab₁ will be referred to as a primary or anti-inflammatory cytokineantibody, and Ab₂ will be referred to as a secondary or anti-Ab₁antibody.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example, fluorescein, rhodamine and auramine.A particular detecting material is anti-rabbit antibody prepared ingoats and conjugated with fluorescein through an isothiocyanate.

The peptides of the inflammatory cytokine or their binding partner(s)can also be labeled with a radioactive element or with an enzyme. Theradioactive label can be detected by any of the currently availablecounting procedures. The preferred isotope may be selected from ¹⁴ C,¹³¹ I, ³ H, ¹²⁵ I and ³⁵ S.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric or gasometric techniques. The enzyme isconjugated to the selected particle by reaction with bridging moleculessuch as carbodiimides, diisocyanates, glutaraldehyde and the like. Manyenzymes which can be used in these procedures are known and can beutilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

A particular assay system developed and utilized in accordance with thepresent invention, is known as a receptor assay. In a receptor assay,the material to be assayed is appropriately labeled and then certaincellular test colonies are inoculated with a quantity of both thelabeled and unlabeled material after which binding studies are conductedto determine the extent to which the labeled material binds to the cellreceptors. In this way, differences in affinity between materials can beascertained.

Accordingly, a purified quantity of the inflammatory cytokine may beradiolabeled, after which binding studies would be carried out using forexample, recently differentiated neutrophils. Solutions would then beprepared that contain various quantities of labeled and unlabeledinflammatory cytokine and unknown cell samples would then be inoculatedand thereafter incubated. The resulting cell monolayers would then bewashed, solubilized and then counted in a gamma counter for a length oftime sufficient to yield a standard error of <5%. These data are thensubjected to Scatchard analysis after which observations and conclusionsregarding material activity can be drawn. While the foregoing protocolis exemplary, it illustrates the manner in which a receptor assay may beperformed and utilized, in the instance where the cellular bindingability of the assayed material may serve as a distinguishingcharacteristic.

In a further embodiment of this invention, commercial test kits suitablefor use by a medical specialist may be prepared to determine thepresence or absence of inflammatory cytokine in a suspected mammal. Forexample, one class of such kits will contain at least a labeledconponent selected from the inflammatory cytokine or its bindingpartner, for instance an antibody specific thereto, and directions, ofcourse, depending upon the method selected, e.g., "competitive","sandwich", "DASP" and the like. The kits may also contain peripheralreagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of thereaction of a mammalian host to invasive stimuli, comprising:

(a) a predetermined amount of at least one labeled immunochemicallyreactive component obtained by the direct or indirect attachment of thepresent inflammatory cytokine or a specific binding partner thereto, toa detectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

(a) a known amount of the inflammatory cytokine as described above (or abinding partner) generally bound to a solid phase to form animmunosorbent, or in the alternative, bound to a suitable tag, or pluralsuch end products, etc. (or their binding partners) one of each;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

In a further variation, the test kit may be prepared and used for thepurposes stated above, which operates according to a predeterminedprotocol (e.g. "competitive", "sandwich", "double antibody", etc.), andcomprises:

(a) a labeled component which has been obtained by coupling theinflammatory cytokine to a detectable label;

(b) one or more additional immunochemical reagents of which at least onereagent is a ligand or an immobilized ligand, which ligand is selectedfrom the group consisting of:

(i) a ligand capable of binding with the labeled component (a);

(ii) a ligand capable of binding with a binding partner of the labeledcomponent (a);

(iii) a ligand capable of binding with at least one of the component(s)to be determined; and

(iv) a ligand capable of binding with at least one of the bindingpartners of at least one of the component(s) to be determined; and

(c) directions for the performance of a protocol for the detectionand/or determination of one or more components of an immunochemicalreaction between the inflammatory cytokine and a specific bindingpartner thereto.

In accordance with the above, an assay system for screening potentialdrugs effective to modulate the synthesis, release, or activity of theinflammatory cytokine may be prepared. In a first procedure, the testdrug could be administered to a stimulated macrophage sample todetermine its effect upon the production of the inflammatory cytokine.In an alternate procedure, the inflammatory cytokine may be introducedinto a cellular test system such as neutrophils, and the prospectivedrug may also be introduced into the cell culture, and the culturethereafter examined to observe any changes in the activity of theinflammatory cytokine, either resulting from the addition of theprospective drug alone, or from the effect of added quantities of theknown inflammatory cytokine.

The primary amino acid sequences shown in FIGS. 10 and 15 are onlyillustrative of the proteins useful in the present invention and similarsequences may result in proteins which have substantially equivalent oraltered activity as compared to that set forth in FIGS. 10 and 15. Thesemodifications may be deliberate, for example, such as modificationsobtained through site-directed mutagenesis, or may be accidental, suchas those obtained through mutations in hosts which are MIP-1 producers.All of these modifications are included in the present invention, aslong as the MIP-1-like activity, as defined above, is retained.Accordingly, the definition of MIP-1 and the term "inflammatorycytokine" as used herein specifically includes protein material withpeptide components having an amino acid sequence substantiallyequivalent to that in FIGS. 10 and 15, either individually or incombination.

Similarly, the term "stimulus" and its plural are intended to apply toinvasive events such infection, as well as conditions caused bywounding, and to idiopathic or spontaneous states that may for exampleoriginate from cellular or metabolic derangements or other causes.

As indicated earlier, the following example sets forth the details ofthe isolation and identification of the present inflammatory cytokine,and the observations noted as to its activity, defining both thedistinctions and similarities in activity between the presentinflammatory cytokine and those factors identified earlier both byapplicants and by others in the field. Naturally, the specific materialsand techniques set forth hereinafter are exemplary only and may vary, sothat the following is presented as illustrative but not restrictive ofthe present invention.

EXAMPLE

The following experiments were conducted to identify and furthercharacterize the inflammatory cytokine of the present invention.Initially, the mediator substance was cultured, the inflammatorycytokine was isolated and its structure then determined, after which abattery of tests were conducted in an effort to elucidate itsactivities, and where possible, to establish or refute identity withother known macrophage-derived factors.

MATERIALS AND METHODS

Materials--Purified, recombinant human cachectin/TNF was obtained fromChiron Corp., Emeryville, Calif. Purified, recombinant human IL-1α wasthe generous gift of Dr. P. Lomedico (Hoffmann LaRoche, Nutley, N.J.).All chemicals were the highest grades available from commercialsuppliers.

Animals--C3H/HeN mice were obtained from Charles River (Kingston, N.Y.).Mice of the endotoxin-resistant C3H/HeJ strain were obtained fromJackson Laboratories (Bar Harbor, Me.).

Cell Culture--The mouse macrophage cell line RAW 264.7 and thecachectin/TNF sensitive cell line L929 were obtained from American TypeCulture Collection (Rockville, Md.) and maintained in RPMI 1640 orDulbecco's modified MEM ((DMEM) GIBCO, Grand Island, N.Y.),respectively. Both media were supplemented with 20 mM Hepes and 10%fetal bovine serum (Hyclone, Logan, Utah.). For the production ofstimulated RAW 264.7 supernatants, cells were grown in 150 mm tissueculture dishes (Falcon) in RPMI plus 10% fetal bovine serum until theyreached confluency. The cells were washed five times in Hanks' balancedsalt solution and the medium was replaced with serum-free RPMIsupplemented with 1 μg/ml of lipopolysaccharide (LPS W, E. coli 0127:B8,Difco, Detroit, Mich.). The cells were incubated at 37° C. for 16-18hours and the supernatants filtered through 0.22 μm filters.

Purification-of-MIP-1--One to five liters of supernatant mediatorsubstance were concentrated 16-40-fold in a DC2 hollow fiberconcentration system with a 10,000 dalton molecular weight cutoff(Amicon Corp., Lexington, Mass.) and diafiltrated against 6 liters of 20mM Tris buffer, pH 8.0, using the same device. Octyl glucoside was addedto the concentrated, diafiltrated supernatant to a final concentrationof 1% (w/v) and the mixture was applied to a Mono Q 10/10 (anionexchange (Pharmacia, Rahway, N.J.)) column previously equilibrated with20 mM Tris buffer, pH 8.0, and connected to a fast protein liquidchromatography (FPLC, Pharmacia) apparatus. A linear gradient of 114 ml(total volume) from 0 to 1 M NaCl in the same buffer (and a flow rate of2 ml/min.) was used for elution.

Samples of each fraction were subjected to polyacrylamide gelelectrophoresis in a denaturing system containing sodium dodecyl sulfate(SDS-PAGE) in 10-15% or 10-18% linear gradient slab gels under reducingconditions. Molecular weight standards (BRL, Inc., Bethesda, Md.) wererun in parallel. Fractions containing MIP-1 eluted in the same region ascachectin/TNF and were easily recognized as a characteristic doublet ofapproximately 8,000 molecular weight.

Peak MIP-1-containing fractions (as assessed by SDS-PAGE and silverstaining) were pooled, concentrated and fractionated on ahigh-performance gel-filtration column (Superose 12; Pharmacia)previously equilibrated with 100 mM ammonium acetate. MIP-1 wasrecovered in the void volume of the column and was greater than 95% pureas judged by SDS-PAGE and silver staining. MIP-1 purified in this mannercontained approximately 0.2 ng LPS/g MIP-1.

Heparin Chromatography--Heparin-conjugated Sepharose (Pharmacia) wasused to assay the ability of MIP-1 to bind heparin. A C10/20 column(Pharmacia) packed with 8 ml of gel was attached to an FPLC apparatusand equilibrated with 20 mM Tris, pH 8.0. Two ml of 12-fold concentratedand diafiltrated RAW 264.7 supernatant (as above) was applied to thecolumn and a linear gradient of 0 to 2 M NaCl in the same buffer wasused for elution.

Chromatofocusing--Eight micrograms of a peak MIP-1-containing fractionfrom Mono Q chromatography was equilibrated in 25 mM bis-Tris plus 10%betaine (w/v), pH 7.1, and applied to a Mono P column previouslyequilibrated in the same buffer. Protein was eluted with a lineargradient of Polybuffer 74 (Pharmacia) (1:10 in double-distilled water)with 10% betaine (pH 4.0), resulting in a descending pH gradient rangingfrom 7 to 4.

Protein Assay--Protein content was measured by an assay (Bradford, M.,ANAL. BIOCH. 72:248-254, (1976)) using BSA as a standard (Bio-Rad.Richmond, Calif.).

Endotoxin Assay--Endotoxin levels were determined using a chromogeniclimulus assay (Whittaker M. A. Bioproducts, Walkersville, Md.) accordingto the instructions of the manufacturer.

Protein Sequencing--Purified MIP-1 was sequenced by the RockefellerUniversity protein sequencing facility. The Dayhoff protein sequencebank was searched for homologous amino acid sequences using the computerprogram d-FAST-P.

In Vivo Inflammatory Activity--Polymorphonuclear leukocyte (PMN)infiltration was evaluated using footpad injections, according toGranstein, R. D., et al., J. CLIN. INVEST. 77:1020-1027 (1986). Briefly,female C3H/HeJ mice (6-12 weeks) were lightly anesthetized withphenobarbital (25 mg/kg body weight, i.p.). Animals were randomized toreceive a subcutaneous footpad injection of 0.05 ml containing 10⁻¹⁰moles N-formylmethionylleucylphenylalanine (fMLP); 10, 100 or 1000 ng ofrecombinant human IL-1α or recombinant human cachectin/TNF; or 1, 10,100, or 1000 ng of murine MIP-1 (purified as above) in RPMI 1640 with0.1% fetal bovine serum. RPMI 1640 with 0.1% fetal bovine serum aloneserved as a control. In some cases, mice received the test substance inone hind-limb foot-pad and the control carrier in the contralateralhindlimb. In other cases, a randomized block design was employed. Micewere sacrificed four hours following injection and hindlimbs were fixedin 10% buffered formalin. Hind-limbs were decalcified, embedded inparaffin and thin-sections of footpads were stained with hematoxylin andeosin.

In Vitro PMN Migration Assays--Heparinized venous blood was obtainedfrom healthy volunteers. Leukocytes containing greater than 95% PMN's asjudged by cell sorter analysis were isolated by Ficoll-Hypaque densitygradient centrifugation and dextran sedimentation (Klempner, M. S., etal., J. CLIN. INVEST. 64:996-1002 (1979)). Residual erythrocytes wereremoved by lysis with hypotonic saline. Cells were resuspended in Gey'sbalanced salt solution (GBSS, pH 7.4) and 2% bovine serum albumin (BSA)to a final concentration of 2.5×10⁶ cells/ml.

In vitro chemokinesis was assayed using a modification of the techniquedescribed by Boyden (Boyden, S. V., J. EXP. MED. 115:453-466 (1962)).Bottom wells of blind well chambers were filled with 25 μl of buffercontaining the test compound, i.e. fMLP (10⁻⁸ M), MIP-1 or buffer alone;and the top wells filled with 45 μl of GBSS/BSA containing 1.1×10⁴PMN's. The two wells were separated by a cellulose nitrate membrane witha 3 μm pore size (SM 11302, Sartorius, Westbury, Conn.). Chambers wereincubated at 37° C. in a humidified 5% CO₂ -95% room air chamber for 45minutes. Membranes were removed and stained according to a previouslydescribed protocol (Hesse, D. G., et al., J. CLIN. INVEST. 73:1078-1085(1984)). The number of PMN's migrating into the membrane was counted forevery 10 μM up to 130 μM using an automated Optomax imaging system(Optomax, Inc., Hollis, N.H.). Migration was quantitated in threerandomly selected fields for each membrane with each sample tested intriplicate. Chemokinesis was defined as the mean distance migrated intothe membrane (expressed as a percent) compared to GBSS alone (0%) andthat of the positive FMLP control (100%).

Chemokinetic data are expressed as percents (mean±standard error of themean (SEM)) of the control chemokinetic response. A one-way analysis ofvariance (ANOVA) was used to compare the response to MIP-1 with that toGBSS alone in the bottom well of the chamber.

Induction of Hydrogen Peroxide Release--The ability of MIP-1 to elicitthe release of H₂ O₂ from adherent human PMN or monocytes was tested bythe method recently described in detail (Nathan, C. F. 1987, J. CLIN.INVEST. (in press)). In brief, PMN and mononuclear leukocytes inheparinized or citrated blood were isolated in Neutrophil IsolationMedium (Packard Instrument Co., Downer's Grove, Ill.), washed and platedseparately at 1.5×10⁴ PMN or 2×10⁵ mononuclear cells per well inflat-bottomed, 6-mm diameter polystyrene tissue culture wells that hadpreviously been coated with fetal bovine serum and extensively washed.The assay mixture contained 2.4 nM scopoletin, 0.5 μg horseradishperoxidase, 1 mM sodium azide and the indicated test agents in a finalvolume of 0.13 ml of Krebs-Ringer phosphate buffer with glucose at 37°C. Loss of fluorescence of scopoletin due to oxidation by H₂ O₂ wasrecorded at 15- or 30-minute intervals in a plate-reading fluorometerand converted to nM H₂ O₂ by microcomputer (de la Harpe, J., et al. , J.IMMUN. METHODS 78:323-336 (1985)).

IL-1 and Cachectin/TNF Bioassays--MIP-1 was assayed for IL-1 activity byits ability to stimulate C3H/HeJ thymocytes to undergo blastogenesis inthe presence of suboptimal quantities of phytohemagglutinin, aspreviously described (Moldawer, L. L., et al., J. IMMUN. 138:4270-4274(1987)).

Cachectin/TNF activity was assayed by its ability to kill actinomycinD-treated L929 murine fibroblasts (Ostrove, J. M., et al., PROC. SOC.EXP. BIOL. MED. 160:354-358 (1979)). Approximately 50,000 L929 cellswere plated in each well of a 96-well plate (Falcon) in DME mediumcontaining 1 μg/ml actinomycin D and increasing quantities of MIP-1.After 14-16 hours, the chromogen(3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT)) wasadded and the cells incubated for an additional 4 hours. Cell viabilitywas assessed by the ability of the cells to reduce the chromogen duringthis time period by a modification of a known procedure (Mosmann T., J.IMMUN. METHODS 65:55-63 (1985)). The medium was aspirated and the cellslysed with 0.04N hydrochloric acid in isopropanol. After addition of onevolume double-distilled water, the extent of chromogen reduction wasmeasured by reading the plates at O.D. 570/690 using an automatedELISA-plate reader and values were compared to a standard cytotoxicitycurve obtained in like manner with recombinant human cachectin/TNF.

Cachectin/TNF was also assayed by the suppression of lipoprotein lipaseon 3T3-L1 cells as previously described (Beutler, B., et al., J. EXP.MED. 161:984-995 (1985)). The ability of MIP-1 to induce cachectin/TNFin primary cultures of macrophages was assessed by eliciting macrophageswith an i.p. injection of two milliliters of sterile thioglycollatebroth (DIFCO) and collecting the cells 4 to 6 days later by peritoneallavage. The cells were washed and resuspended in serum-free RPMI andplated at 10⁶ cells/well in 24-well tissue culture plates. Testsubstances (LPS, 0.0001-1 μg/ml; MIP-1, 1 μg/ml) were added and thecells incubated at 37° C. for 18 hours. The cell-free supernatants werecollected and assayed for cachectin/TNF activity by cytotoxicity on L929cells as described above.

Purification of MIP-1α and MIP-1β: MIP-1 containing fractions from highperformance gel filtration chromatography over Superose 12 were pooled,concentrated using a PM-10 membrane in a stirred cell (Amicon Corp.,Danvers, Mass.), and diafiltrated against 0.01M sodium phosphate buffer,pH 6.4. Two components of MIP-1 were then resolved bySDS-hydroxylapatite chromatography according to a known procedure (Moss,B. and E. N. Rosenblum., J. BIOL. CHEM., 247:5194, (1972)). In brief, a500 μg aliquot of MIP-1 was equilibrated in 0.01 M sodium phosphatebuffer, pH 6.4, containing 1% SDS and 1% mercaptoethanol and placed in aboiling water bath for 2 minutes. The sample was immediately diluted10-fold with 0.01 M sodium phosphate buffer (pH 6.4) containing 0.1% SDSand 1.0 mM DTT (Buffer A), applied directly to a hydroxylapatite column(Bio Gel HPHT, Bio-Rad, Richmond, Calif.) pre-equilibrated in Buffer A.A 25 ml linear gradient from 0.01 M to 0.35 M sodium phosphate buffer(pH 6.4) containing 0.1% SDS and 1.0 mM DTT was used for elution.

Protein Sequencing: N-terminal amino acid sequence analysis of MIP-1αand MIP-1β were performed on an Applied Biosystems gas phase sequenator.The Dayhoff protein sequence bank was searched for homologous amino acidsequences using the computer program d-FAST-P.

Hydropathicity Plots: The hydropathicity plots of MIP-1α and MIP-1β werecalculated by a modification of a known algorithm (Hopp, T. P. and K. R.Woods., PROC. NATL. ACAD. SCI. U.S.A. 78:3824 (1981)).

Construction of cDNA Library: A cDNA library from LPS-stimulated RAW264.7 cells was obtained as described previously (Davatelis, G., S. D.Wolpe, C. Luedke, P. Tekamp-Olson, J. Merryweather, K. Hermsen, C.Gallegos, D. Coit,.and A. Cerami, J. EXP. MED. In press (1988)). Inbrief, confluent monolayers of RAW 264.7 cells were washed five times inHBSS and covered with serum-free RPMI 1640 culture medium containing 1.0μg/ml LPS. After incubation at 37° C. for 2 hours, total RNA wasextracted into 6M guanidinium thiocyante (Ullrich, A., J. Shine, J.Chirgwin, R. Pictet, E. Tischer, W. J. Rutter, and H. M. Goodman,SCIENCE 196:1313 (1977)). Poly(A)+ RNA was then isolated by two cyclesof oligo(dT)-cellulose chromatography according to a modification of aknown procedure (Maniatis, T., E. F. Fritsch, and J. Sambrook,"Molecular Cloning. A Laboratory Manual." Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. p. 197 (1982)). Double-stranded cDNA wasprepared from the poly(A)+ selected RNA according to a known procedure(Gubler, U., and B. J. Hoffman, GENE. 25:263 (1983)). Internal Eco R1sites were methylated, Eco R1 linkers added, and the cDNA inserted intothe Eco R1 sites of bacteriophage lambda gt10 (Huynh, T. V., R. A.Young, and R. W. Davis, DNA Cloning: A Practical Approach, D. M. Gloves,ed., IRL Press, Oxford. 149 (1985)).

Construction of the Probe Pools: Two oligonucleotide probe poolscorresponding to the "major" sequence were synthesized according to aknown procedure against amino acids #22-30 of a partial amino-terminalsequence. This portion of the polypeptide was selected because of itslower degeneracy in the codon dictionary when compared to the remainderof the sequence. The resulting probe pools are two 512-fold degeneratepools of 26 nucleotides in length.

An oligonucleotide probe pool corresponding to the "minor" amino acids2-8 of a partial N-terminal sequence of MIP-1β were synthesized asdescribed by a modification of a known method (Warner, B. D., M. E.Warner, G. A. Karns, L. Ku, S. Brown-Shimer, and M. S. Urdea, DNA. 3:401(1984). This particular sequence was chosen because it was the region ofleast apparent homology between MIP-1α (major) and MIP-1β (minor)sequences.

Screening of the Library: In regard to MIP-1α, nitrocellulose filterlifts of a low-density plating (5×10³ pfu/plate) of the library werehybridized using the synthetic probe pool that had been 5'-end labeledwith ³² P-ATP (New England Nuclear, Boston, MA). Following thehybridization, the lifts were washed using the method of Wood et al.(10). After several rounds of screening, 18 recombinant phage cloneswere isolated and grown in bulk for DNA isolation. In regard to MIP-1β,duplicate nitrocellulose filter lifts of the plated library (4×10⁵plaques) were hybridized overnight at 42° C. in 4×SSC, 2× Denhardt'ssolution, 40 mM sodium phosphate buffer, pH 7.0, 0.3 mg/ml sonicatedsalmon sperm DNA, 0.1% SDS and 2.5-5×10⁴ cpm/ml/degeneracy of the ³²P-ATP 5' end-labeled synthetic oligonucleotide probe pool. Followinghybridization, the filters were washed; the final washes were done underconditions of moderate stringency: 2×SSC, 0.1% SDS at 50° C. Plaqueswhich were positive on duplicate filters were subjected to a secondround of low density plating and screening. In this way two independentpositive phage clones were isolated from which DNA was prepared forfurther analysis.

DNA Sequence Analysis: The cDNA inserts to be analyzed were subclonedinto M13 phage vectors and DNA sequencing was performed by thedideoxy-chain termination method of Sanger et al. (F. Sanger and S.Nicklen, R. Coulson, PROC. NATL. ACAD. SCI. U.S.A. 74:5463 (1977)).

Blot Hybridization Analysis--Northern blot hybridization was performedby an adaptation of a known method (Lehrach, H., D. Diamond, J. M.Wozney, and H. Boedtker, BIOCHEMISTRY. 16:4743 (1977)). Total RNA ofLPS-stimulated and nonstimulated RAW 264.7 cells were electrophoresedthrough 1.2% agarose gels and transferred to nitrocellulose filters.

Primer Extension--The synthetic oligonucleotide primer was end labeledusing [τ-³² P]ATP (3,000 Ci/mmol, Amersham Corp., Arlington Heights,Ill.) and the T4 polynucleotide kinase. The primer extension method wasa modification of a known method (Walker, M. D., T. Edlund, A. M.Boulet, and W. J. Rutter, NATURE LOND. 306:557 (1983)).

RESULTS

Purification of MIP-1--When supernatants of stimulated RAW 264.7 cellswere fractionated by Mono Q (anion exchange) chromatography, MIP-1 wasapparent as a distinctive doublet of about 8,000 daltons on SDS-PAGEafter silver staining (FIG. 1). MIP-1 reproducibly eluted within afraction or two of cachectin/TNF at approximately 0.37 M NaCl andappeared to be produced in approximately the same quantities as judgedby silver-staining.

Chromatofocusing revealed that Mono Q-purified MIP-1 eluted at aslightly more acidic pH than cachectin/TNF. This corresponded to a pI of4.6 (not shown).

In order to further purify MIP-1, advantage was taken of its tendency toaggregate. Aggregation of MIP-1 was observed to occur during theconcentration of the crude material and before diafiltration orfractionation on Mono Q (data not shown). When fractionated by gelfiltration in phosphate-buffered saline, MIP-1 formed multimers ofvarious molecular weights ranging from approximately 20,000 daltons tomaterial eluting in the void volume (≧2×10⁶ daltons; data not shown). In100 mM ammonium acetate this tendency was exaggerated and the majorityof the protein eluted in high molecular weight fractions (FIG. 2). Underthese conditions, MIP-1 of greater than 95% purity, as judged bySDS-PAGE and silver staining, was obtained.

Partial N-terminal amino acid sequence data of purified MIP-1 (FIG. 3)showed a single major sequence although sequences of two separatebatches showed consistent minor amino acids at several specificpositions in the N-terminal region. Computer-based analysis of thissequence data revealed no significant homology with any previouslydescribed protein.

Affinity of MIP-1 for Heyarin--During the purification of MIP-1, itsaffinity for heparin (FIG. 4) was noted. When 2 ml of 12-foldconcentrated RAW 264.7 supernatant were applied to a column and elutedwith a linear gradient of 0 to 2 M NaCl, MIP-1 was one of two majorproteins detectable by SDS-PAGE and silver staining, eluting atapproximately 0.7 M NaCl.

Determination of Biological Activities--At concentrations as high as 20gg/ml, purified MIP-1 did not stimulate blastogenesis of C3H/HeJthymocytes. Recombinant human IL-1, on the other hand, was active inthis assay at concentrations as low as 10 pg/ml (data not shown).Similarly, purified MIP-1 did not kill L929 cells in the presence ofactinomycin D even at concentrations of 1 μg/ml whereas recombinanthuman cachectin/TNF was able to induce killing at concentrations as lowas 15 pg/ml (data not shown). Further, purified MIP-1 did not inducedown-regulation of lipoprotein lipase in 3T3-L1 cells (data not shown).At 1 μg/ml, purified MIP-1 did not induce cachectin/TNF production byprimary thioglycollate-elicited mouse macrophages in the presence of 10μg/ml polymyxin B (data not shown).

Although free of the above-mentioned IL-1- and cachectin/TNF-likeactivities, MIP-1 did induce a localized inflammatory response at fourhours when injected subcutaneously into the footpads of C3H/HeJ mice.Maximal inflammation occurred when 100 ng of MIP-1 were administered andwas characterized primarily by PMN leucocyte infiltration (FIG. 5, plateA). The control response to an injection of carrier is shown in plate B.The degree of neutrophil infiltration seen with MIP-1 was not as markedas that seen when 10⁻¹⁰ moles of fMLP were administered (FIG. 5, plateC). However, the degree of neutrophil infiltration was comparable tothat observed with 10 ng of recombinant cachectin/TNF. Recombinant humanIL-1 elicited no inflammatory 25 responses at four hours whenadministered at these doses (data not shown).

MIP-1 induced chemokinesis in human neutrophils in vitro. Data presentedin FIG. 6 represent the mean per cent of the control migratory responseof five experiments. Values are presented as the percent increases inneutrophil migration relative to the negative GBSS (0%) and positivefMLP 10⁻⁸ M controls (100%). At concentrations equal to or greater than100 ng/ml, MIP-1 elicited a significant increase in neutrophil migration(p<0.01 by ANOVA). Inclusion of polymyxin B (10 μg/ml) had no effect onMIP-1-induced chemokinesis. Further, LPS at concentrations of 10-1000ng/ml was not active in this assay (data not shown).

Recombinant cachectin/TNF, but not rIL-1α, triggers a delayed butsignificant respiratory burst in human PMN, provided the cells areadherent to a surface coated with serum or extracellular matrix proteins(Nathan, C. F. 1987, J. CLIN. INVEST. (in press)). Similarly, MIP-1at >1 μg/ml triggered adherent PMN to release H₂ O₂ in four experiments,one of which is illustrated in FIG. 7. Compared to cachectin/TNF testedin parallel cultures, the response to MIP-1 was more delayed (60 minutelag versus 15 minute lag for cachectin/TNF), and the maximal sustainedrate was lower (1.2 nmol/min per 10⁶ PMN versus 3.0 for cachectin/TNFand 2.1 for PMA). However, the duration of the response was greater (2.5hours compared to 1 hour for cachectin/TNF) and thus, the total amountsof H₂ O₂ released were similar. Because MIP-1 binds heparin, experimentswere also performed with PMN isolated from citrated rather thanheparinized blood and gave similar results.

Neither MIP-1 (not shown) nor cachectin/TNF (Nathan, C. F. 1987, J.CLIN. INVEST. (in press)) triggered H₂ 0₂ release from monocytes.

DISCUSSION

The data presented above indicate that, while the inflammatory cytokineMIP-1 bears no significant sequence homology to any previously describedprotein, it shares some of the overlapping properties typical ofinflammatory mediators such as cachectin/TNF and IL-1.

MIP-1 was isolated on the basis of its interesting physical properties.As indicated earlier, although MIP-1 migrates as a doublet of about8,000 daltons on SDS-PAGE, it readily forms high molecular weightaggregates in excess of 2×10⁶ daltons as judged by gel filtration.Partial amino acid sequence data show one "major" sequence with "minor"substitutions at several positions.

The binding of MIP-1 to heparin under conditions where the protein isanionic suggests a specific interaction. This is further emphasized bythe observation that MIP-1 is one of two major macrophage-secretedproteins that bind to heparin at high salt concentrations. It ispossible that MIP-1 may play a role in the coordination of theinflammatory activities of macrophages, mast cells and neutrophils.MIP-1 may also interact with basement membrane proteoglycans duringinflammation.

The findings presented here are consistent with the suggestion thateither cachectin/TNF or MIP-1 are capable of inducing an inflammatoryresponse. Cachectin/TNF has previously been shown to induce neutrophilchemokinesis (Figari, I. S., et al. 1987, BLOOD (in press); and Ming, W.J., et al., J. IMMUN. 138:1469-1474 (1987)) as well as activation(Shalaby, M. R., J. IMMUN. 135:2069-2073 (1985); and Tsujimoto, M., etal., BIOCH. BIOPHYS.

RES. COMMUN. 137:1094-1100 (1986)). In the present study MIP-1 was shownalso to be capable of inducing neutrophil chemokinesis. In addition, atthe doses used here, cachectin/TNF and MIP-1 each elicited similardegrees of inflammation in vivo. Although others have shown thatrecombinant IL-1 can induce an inflammatory reaction in vivo (Granstein,R. D., et al., J. CLIN. INVEST. 77:1020-1027 (1986)), no such effect wasfound here; it is possible that the recombinant human IL-1α used here isless active in this regard than the murine IL-1α used in publishedexperiments (Granstein, R. D., et al., J. CLIN. INVEST. 77:1020-1027(1986)).

It is unlikely that the effects of MIP-1 administration are due tocachectin/TNF contamination as there was no cachectin/TNF bioactivitydetected by L929 cytotoxicity assay at the doses used. Further, MIP-1did not induce primary thioglycollate-elicited macrophages to producecachectin/TNF. Endotoxin contamination was ruled out as an explanationfor the chemokinetic effect, as chemokinesis was not affected by thepresence of polymyxin B; and LPS itself, at concentrations greater thanwere present in the MIP-1 assays, had no chemokinetic effect.

The inflammation induced by MIP-1 was observed in mice of theendotoxin-resistant C3H/HeJ strain using preparations with low levels ofendotoxin contamination (0.2 ng LPS/μg MIP-1).

As described previously, native murine MIP-1 is isolated as a doubletwith nearly identical subunit molecular weights of approximately 8000daltons. Although two MIP-1 components are separated to some extent bySDS-PAGE, their electrophoretic mobilities are so similar thatpreparative SDS-PAGE appeared an impractical means of purification.Native MIP-1 (doublet) was subjected to SDS-hydroxylapatitechromatography, a technique which has been used successfully to separateprotein subunits of similar electrophoretic mobility (B. Moss and E. N.Rosenblum, J. BIOL. CHEM. 247:5194 (1972)). As can be seen in FIG. 13,two distinct protein peaks are observed following fractionation ofnative MIP-1 on a hydroxylapatite column in the presence of SDS. Thefirst peak elutes at 0.24 M sodium phosphate and the second peak elutesat 0.27 M sodium phosphate. SDS-PAGE analysis of the column fractionsrevealed that the first peak corresponded to the lower molecular weightband of MIP-1 now referred to as MIP-1α, while the second peakcorresponded to the higher molecular weight band referred to as MIP-1β.Using this technique, each component was obtained in pure form (>95% asjudged by SDS-PAGE) for N-terminal amino acid sequence analysis.

To elucidate the molecular structure of murine MIP-1α, a cDNA clone wasisolated containing the sequence coding for MIP-1α. As a first step, themouse macrophage cell line RAW 264.7 was stimulated with LPS. Since RAW264.7 cells have been shown to be a source of MIP-1α protein after LPSstimulation, the MIP-1α mRNA was expected to be highly reiterated inthese LPS-induced cells. Poly(A) +RNA was prepared from total RNA by twocycles of lambda oligo-dT-chromatography and a cDNA library wasconstructed in lambda gt10. The cloning efficiency was 10⁶ clones/μg ofpoly(A) +RNA. The library was amplified and shown to contain inserts ofgreater than 1,000 bp in above 60% of recombination plaques.Nitrocellulose filter lifts of a low-density plating of the library werescreened using two synthetic oligonucleotide pools that were based onthe partial NH₂ -terminal amino acid sequence of purified MIP-1 (S. D.Wolpe, G. Davatelis, B. Sherry, B. Beutler, D. G. Hesse, H. T. Nguyen,L. L. Moldawer, C. F. Nathan, S. F. Lowrey, and A. Cerami, J. EXP. MED.167:570 (1987)). Each pool consisted of a 512-fold degeneracy pool 26nucleotides in length (FIG. 9).

After the initial library screening, positive plaques were streaked ontofresh bacterial lawns and a secondary screening was performed bydifferential plaque hybridization. Replicate lifts of the secondarystreaks were hybridized to either ³² P-labeled pool #1 or pool #2. Sincethe melting temperature (T) of DNA/DNA hybrids can be approximated bythe empirical formula: T=16.6(log[Na+]+0.41(%[G+C])+81.5-500/number ofbp in homology, one of the probe pools was effectively eliminatedthrough the differential melting temperatures of the hybrids based on a26-bp homology. By using a tetramethylammonium chloride washingtechnique (W. Wood, I. J. Gitschier, L. A. Laskey, and R. M. Lawn, PROC.NATL. ACAD. SCI. USA. 82:1585 (1985)), which abolishes the preferentialmelting of A-T vs. G-C base pairs, the melting temperature (Tm) becomesdependent simply on the length of the hybrid.

After several rounds of differential hybridization, probe pool #1yielded 18 recombinant phage clones out of 10⁴ screened that hybridizedunder maximally stringent conditions for MIP-1α. All of the plaques werepurified and DNA was prepared from each. The recombinant phage clone 52appeared to contain the largest cDNA Eco-RI insert of about 750 bp andwas chosen for further characterization. The complete nucleotidesequence of cDNA clone 52, as well as 262 bp of 5' sequence of another,partially overlapping clone, 32, have been determined and are shown inFIG. 10. The latter clone, which was isolated in a later screening, hada smaller Eco RI insert than clone 52, but a larger 5'-end fragment andtherefore presumably less poly(A) tail. The MIP-1α nucleotide sequenceof 763 bp predicts a single open reading frame starting at nucleotide 2.The mature protein sequence, starting at position one, is 69 amino acidsin length and comprises the "major" sequence previously defined by NH₂-terminal analysis of the purified MIP-1 protein (S. D. Wolpe, G.Davatelis, B. Sherry, B. Beutler, D. G. Hesse, H. T. Nguyen, L. L.Moldawer, C. F. Nathan, S. F. Lowrey, and A. Cerami, J. EXP. MED.167:570 (1987)).

The first methionine present in the sequence is found at position -23.This is postulated to be the initiating methionine for the MIP-1αprecursor based on the following observations. Structural analysis ofthe putative presequence (-23 to -1) indicates that it has featurescharacteristic of typical signal sequences (i.e., α-helix and ahydrophobic core (D. Perlman and H. O. Halvorson, J. MOLE. BIOL.,167:391(1983)). The predicted initiating ATG has a purine at relativeposition -3, which has been shown (M. Kozak, CELL. 44:283 (1986)) tohave a dominant effect on translation imitation efficiency. Furthermore,in a survey of the frequency of A,C,T,G around the translation startsite of 699 vertebrate mRNAs, 97% had a purine at position -3, 61%having an A at that position (M. Kozak, NUCLEIC ACIDS RES. 15:8125(1987)). NA clone. oligonucleotide primer (FIG. 10) was hybridized toLPS-stimulated RAW 264.7 poly(A) +RNA and elongated with reversetranscriptase. After hybridization, an extended primer of 98±2nucleotides was obtained (data not shown). After subtracting out theprimer length of 25 nucleotides and the sequence 5' to the primer we hadpreviously determined (61 nucleotides), one can conclude that the knownsequence is 10-14 nucleotides short of a full-length cDNA. While it ispossible than an in-frame AUG is present in this cloned region, it seemshighly unlikely given that only 14 of 346 sequenced vertebrate mRNAshave 5' noncoding sequences less than 19 nucleotides in length ((M.Kozak, NUCLEIC ACIDS RES. 15:8125 (1987)). It can then be estimated thatthe 5' untranslated sequence is about 82 nucleotides, well within the20-100 nucleotide length of most vertebrate 5' noncoding regionssequenced to date.

The proposed pre-MIP-1α is 92 amino acids in length. There are noconsensus Asn-X-Ser,Thr sites for N-linked glycosylation evident in themolecule. There are 7 cysteines, 3 in the presequence and 4 in themature sequence. The codon usage of the putative pre-MIP-1α agrees wellwith that determined for 66 other sequenced murine genes (T. Marayama,T. Gojobori, S. Aota, and T. Ikemura, NUCLEIC ACIDS RES. 14(Suppl):rl15(1986)). The peptide has no significant sequence similarity to anyprotein as defined to date by the d-fast-P program homology search (D.J. Lipman and W. R. Pearson, SCIENCE, 227:1435, (1985)) of the Dayhoffprotein data base. The DNA sequence was also compared against GenBankgenetic sequence data with a similar negative result.

In the 3'-untranslated region, there is a single consensuspolyadenylation site at bp 711 to 716. There are also 4 sequences thathave only one mismatch to a known cytokine consensus 3'-untranslatedsequence (Copending application Ser. No. 017,360, filed Feb. 24, 1987,the disclosure of which is incorporated herein by reference. See also,D. Caput, B.

Beutler, K. Hartog, R. Thayer, S. Brown-Shimer, and A. Cerami, PROC.NATL. ACAD. SCI, 83:1670 (1986)). The 3'-untranslated consensus cytokinesequence (TATT)_(n) is also present (R. Reeves, A. G. Spies, M. S.Nissen, L. D. Buch, A. D. Weinberg, P. J. Barr, N. S. Maguuson and J. A.Maguuson, PROC. NATL. ACAD. SCI. USA 83:3228 (1986). When n=2 and onemismatch is allowed, four of these sequences are found. There is anoverlap in three of these between the sequence defined by Caput et al.and that defined by Reeves et al.

Since MIP-1 is an inducible protein, the expression of murine MIP-1αmRNA by Northern blot hybridization in RAW 264.7 cellular 30 DNA wasalso studied. As shown in FIG. 11, total RNA from LPS-induced cellsexhibited a positive hybridization band with an estimated size of 800bp, while total RNA from uninduced cells showed very little of apositive signal at that or any other size. In a time-course study of theinduction of murine MIP-1α mRNA by endotoxin in these same cells (FIG.12), murine MIP-1 mRNA is detected within 1 hour after LPS stimulationand peaks between 8-16 hours after LPS stimulation. This time coursediffers from the appearance of either murine TNF-α/cachectin or IL-1αmRNAs when their respective plasmid probes were hybridized to the sameblot.

An mRNA produced by human tonsillar lymphocytes in response to mitogenwas reported by Obaru, M. Fukuda, S. Maeda, and K. Shimada, J. BIOCHEM,99:885 (1986). While the sequence is not listed in the Dayhoff proteindata base or the GenBank genetic sequence data base, there is a 75.3%amino acid sequence similarity between that protein and murine MIP-1α.The relationship between these proteins is not known.

Results of N-terminal sequence analysis of the higher molecular weightcomponent of MIP-1 (now referred to as MIP-1β) is shown in FIG. 14B. The"minor" residues observed at positions 3 and 7 in the originalN-terminal amino acid sequence correspond to amino acid differencesbetween MIP-1β and MIP-1α. In FIG. 14C the original N-terminal aminoacid consensus sequence for native MIP-1 is given for comparison.

A cDNA clone containing the coding sequence for MIP-1β was isolated andcharacterized. To accomplish this, a cDNA library from LPS-stimulatedRAW 264.7 cells was prepared. The oligonucleotide probe pool used toscreen the cDNA library was generated against the sequence underlined inFIG. 14B. Three of these seven MIP-1β amino acids differ from thecorresponding sequence of MIP-1β. The probe pool utilized for screeningthe library was

                       TC*  *                                                            5'-CCXATGGGX  CGACCCCXCC-3'.                                                              AG                                                     

The third position choices were made based on the codon usage reportedfor cloned murine genes (F. Sanger, S. Nicklen, and R. Coulson, PROC.NATL. ACAD. SCI. (U.S.A.) 74:5463, (1977) and the codon usage of MIP-1α.

Utilizing the labeled probe pool, the library was screened underconditions of moderate stringency. Two independent clones were isolatedout of 4×10⁵ recombinant phage plaques screened. This was a considerablylower frequency than expected. Upon DNA sequencing of the isolatedclones, it was found that the choice of C for the third position of theSer5 codon was incorrect.

The correct choice was actually a T thereby resulting in no perfectmatch of any of the probe pool sequences to the actual MIP-1β sequence.Thus, MIP-1β sequence representation in the library is likely to beunderestimated based on hybridization with the above probe pool.Subsequent screening of the library with unique oligonucleotide probesspecific for comparable regions of MIP-1α and MIP-1β indicate thatMIP-1β-specific sequences are 5-fold less abundant than MIP-1αsequences.

Following plaque purification, insert DNA (≈700 bp) from the twopositive recombinant phage was shown to cross-hybridize to the MIP-1αcDNA probe. Insert cDNA was isolated from each recombinant phagepopulation and cloned into ml3 from which the complete nucleotidesequence was determined. The two nucleotide sequences differed only inthe length of 5' untranslated sequence present. The longer nucleotidesequence is presented in FIG. 15.

The MIP-1β cDNA is 650 base pairs (which by primer extension analysis isno more than 11-12 nucleotides short of a complete cDNA sequence). Thenucleotide sequence of MIP-1β contains a single open reading framebeginning with the first ATG codon encountered at the 5' end of thesequence (nucleotides +62 to +64) after an in-frame stop codon. Themethionine specified by this codon defines the start of a putativesignal sequence (residues -23 and -1) with characteristic featuresincluding an α-helix and a centrally located hydrophobic region (D.Perlman, and H. O. Halvorson, J. MOL. BIOL., 167:391 (1983)). Thesequence surrounding this ATG codon conforms to the consensus sequenceshared by many mRNAs of higher eukaryotes (M. Kozak, NATURE, LONDON,308:241 (1984)).

The TGA termination triplet is located 91 codons downstream of theinitiating codon. The 3' untranslated region of MIP-1β is comprised of315 nucleotides and contains the hexanucleotide AATAAA (nucleotides +283to +288) which precedes the site of polyadenylation in many eukaryoticmRNAs (N.J. Proudfoot and G.

G. Brownlee, NATURE, LONDON 263:211 (1976)).

There is a single sequence at nucleotides +174 to +181 which preciselymatches a known cytokine 3' untranslated consensus sequence (TTATTTAT)that is characteristic of many immunomodulatory proteins. In addition,there are two additional sequences present that have a single mismatchto this consensus sequence (nucleotides +165 to +175 and nucleotides+185 to +192.

The mature protein sequence, starting at position 1, is 69 amino acidsin length. The molecular weight of the mature protein as determined fromits cDNA clone is 7832 daltons which agrees with the molecular weightprediceted by SDS-PAGE. The amino acid sequence predicted from thecloned DNA sequence is consistent with the first 13 amino acids obtainedfrom N-terminal amino acid sequencing of purified native MIP-1β.

MIP-1β has a single N-linked glycosylation site present in the matureprotein at amino acids 53 to 55 (Asn-Pro-Ser). Whether native MIP-1 isactually glycosylated at this position has not yet been determined, butit is well recognized that proline at position X in the consensusAsn-X-Ser,Thr signal for N-linked glycosylation results in impaired orno glycosylation at that site (I. Moronen, and E. Karajalainen, BIOCHEM.BIOPHYS. ACTA., 788:364 (1984)).

Hydropathicity plots for MIP-1α and MIP-1β which estimate the predictedpolarity profiles of the two proteins are shown in FIG. 16. It is clearfrom FIG. 16 that the local polarity distributions within MIP-1α andMIP-1β are strikingly similar.

The entire nucleotide sequence of the murine MIP-1β cDNA was compared tosequences in the Genbank nucleotide database (rodent, primate, othermammals and other vertebrate libraries) as well as to the MIP-1α cDNAsequence (G. Davatelis, S. D. Wolpe, C. Luedke, P. Tekamp-Olson, J.Merryweather, K. Hermsen, C. Gallegos, D. Coit, and A. Cerami, J. EXP.MED., 167:570 (1988)). MIP-1β and MIP-1α cDNAs are 56.7% identical. Theonly significant homology found in Genbank was the human LD78 cDNA cloneisolated from a T cell lymphocyte cDNA library on the basis that itsmRNA was induced by either the tumor promoter PMA or byphytohemagglutinin (K. Obaru, M. Fukada, S. Maeda, and K. Shimada, J.BIOCHEM., 99:885 (1986)). Murine MIP-1β cDNA showed 57.1% identity in a592 nucleotide overlap to human LD78 cDNA, murine MIP-1α cDNA showedeven higher homology, 69% identity in a 746 nucleotide overlap.

When the predicted amino acid sequence of MIP-1β was tested for homologyto sequences in the Dayhoff data base using the p-Fast-D computerhomology algorithm, no strikingly high homology was found. However,comparison of the predicted protein sequences of murine MIP-1β, murineMIP-1α, human LD78 as well as the recently reported predicted sequencesfor the PDGF, IL-1, a murine cytokine (JE) inducible by double-strandedRNA (P. R. Burd, G. J. Freeman, S. D. Wilson, M. Berman, R. DeKruyff, P.R. Billings, and M. E. Dorf, J. IMMUNOL., 139:3126 (1987) and a murineT-cell activation protein (TCA3) (B. F. Rollins, E. D. Morrison, and C.D. Stiles, PROC. NATL. ACAD. SCI. U.S.A., 85:3738 (1988)) indicates thatthese proteins are noticeably homologous. The deduced amino acidsequence of MIP-1β shows 59.8% identify to that of MIP-1α and 58.7%,38.9% and 21.9% identity to the predicted amino acid sequences of LD78,JE and TCA3 respectively. Common to all these sequences is the presenceof four conserved cysteines in each of the mature peptide sequences.Interestingly, the predicted amino acid sequence of murine MIP-1α ismore homologous to that of human LD78 (75.3% identify) than it is to thepredicted amino acid sequence of murine MIP-1β (59.8% identity). Thus,these proteins share similarities in sequence that appear to define afamily of peptides which may be involved in inflammatory and/or immuneresponses.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended Claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

What is claimed is:
 1. A method for measuring the binding sites for aninflammatory cytokine having an apparent component molecular weight ofabout 8000 Daltons as determined by SDS-PAGE is anionic underphysiological conditions, and capable of binding to heparin, inducinglocalized inflammation characterized by polymorphonuclear cellinfiltration when administered subcutaneously by inducing in vitropolymorphonuclear cell chemokinesis, while lacking the ability tosuppress the activity of the anabolic enzyme lipoprotein lipase, causethe cytotoxicity of cachectin/TNF-sensitive cells, stimulate theblastogenesis of endotoxin-resistant C3H/HeJ thymocytes, or induce theproduction of cachectin/TNF by primary thioglycolate-elicited mousemacrophage cells, wherein the binding sites for said inflammatorycytokine are measured by:A. placing a detectible label on saidinflammatory cytokine; B. placing the labeled inflammatory cytokine incontact with a biological sample from a mammal in which binding sitesfor said cytokine are suspected; and C. measuring said binding sites forsaid cytokine in the biological sample.
 2. The inflammatory cytokine ofclaim 1 wherein said inflammatory cytokine has the amino acid sequenceas set forth in FIG.
 10. 3. The inflammatory cytokine of claim 1 whereinsaid inflammatory cytokine has the amino acid sequence as set forth inFIG.
 15. 4. The inflammatory cytokine of claim 1 wherein saidinflammatory cytokine is a peptide doublet having the amino acidsequences as set forth in FIGS. 10 and
 15. 5. The inflammatory cytokineof claim 1 wherein said cytokine is anionic under physiologicalconditions and binds to heparin at high salt concentrations, and tendsto form aggregates of high molecular weight greater than about 10⁶Daltons in low salt buffers.
 6. The inflammatory cytokine of claim 1wherein said cytokine is capable of inducing fever in rabbits aidinducing superoxide formation or respiratory burst in human neutrophilsin vitro.